The tartrate resistant acid phosphatases (TRACP: EC3.1.3.2) in the serum are acid phosphatases which are mostly derived from osteoclasts. The measurement of acid phosphatases is useful as an index for evaluating the function of osteoclasts, and the acid phosphatases therefore attract attention as a bone resorption marker (Non Patent Literature 1). In the meantime, the acid phosphatase in the serum is analyzed by polyacrylamide gel electrophoresis to provide 6 bands (0 to 5 from an original point). Of these bands, the substance of the 5th band exhibits resistance to tartrate and thus called Band 5 tartrate resistant acid phosphatase (TRACP5). TRACP5 is further electrophoretically divided into TRACP 5a, which often binds to sialic acid of a sugar chain and TRACP 5b, which does not virtually bind to sialic acid. TRACP 5a is an enzyme derived from platelets and others and exhibit a constant blood level; whereas, the blood level of TRACP 5b alone varies in accordance with bone resorption. It is therefore considered that TRACP 5b is a tartrate resistant acid phosphatase itself derived from osteoclasts (Patent Literature 1).
Note that, also in Clinical Chemistry (Non Patent Literature 2), it is recommended that ACP derived from osteoclasts is abbreviated as TRACP-5b. Thus, in the specification, ACP, which is derived from osteoclasts and used as an index of bone resorption, is referred to as TRACP-5b. Tartrate resistant acid phosphatase derived from osteoclasts and tartrate resistant acid phosphatase 5b, which are regarded as synonym, are expressed as TRACP-5b.
Conventional activity measurement methods for obtaining TRACP (acid phosphatase) activity used as an index representing the activity of osteoclasts have a problem in specificity, sensitivity, intricate measurement process and measurement time.
Generally in measurement of TRACP-5b by an activity measurement method, the activity of the enzyme is obtained by subjecting a synthetic substrate, i.e., a phosphoric acid ester, to an enzymatic reaction in the presence of tartaric acid and colorimetrically determining the amount of resultant reaction product (alcohol or phenol). At this time, since tartaric acid inhibits prostate-derived acid phosphatase, the activity of the remaining acid phosphatase is measured by using a substrate to obtain the TRACP activity, which is regarded as TRACP-5b activity. However, erythrocyte-derived and platelet-derived tartrate resistant acid phosphatases present in a specimen except osteoclast-derived tartrate resistant acid phosphatase are also measured herein. Thus, specificity is a problem herein. To improve this method, a method of pretreating a solution obtained by diluting the serum 5 fold by incubating it at 37° C. for one hour and thereafter and measuring the remaining TRACP activity by using p-nitrophenyl phosphate (pNPP) as a substrate, in the presence of tartaric acid (Non Patent Literature 3 and Non Patent Literature 4) is known. In this method, the effect of the erythrocyte-derived acid phosphatase can be avoided, however, the effect of the platelet-derived acid phosphatase cannot be eliminated. The present inventors have reported, as a method for further more specifically measuring the activity, a TRACP-5b measurement method (Patent Literature 2), which uses difference in sensitivity to fluorine between activities of TRACP-5b and erythrocyte/platelet-derived tartrate resistant acid phosphatases. However, the effects of erythrocyte/platelet-derived tartrate resistant acid phosphatases are avoided but the effect of TRACP-5a cannot be eliminated. In addition, TRACP-5b activity is obtained by subtracting the activity not inhibited in the presence of fluorine from the total activity of the tartrate resistant acid phosphatases. Thus, accuracy is another point of improvement to be desired. Furthermore, a method of more specifically measuring TRACP-5b activity was reported to be established by using the method employing fluorine in combination with a TRACP-5a inhibitor (Patent Literature 3). However, although specificity is improved compared to the method employing fluorine alone, osteoclast-derived TRACP-5b activity is obtained computationally by subtraction. For the reason, accuracy still remains as a problem.
Human TRACP-5α and TRACP-5b, both of which are isoforms derived from human ACP5 gene product constituted of 325 amino acids (SEQ. ID No. 1). TRACY-5a is formed by removing a secretory signal sequence consisting of 1st to 21st amino acids; whereas, TRACP-5b is formed by further removing a peptide consisting of the 162nd to 181st amino acids by cathepsin K and connecting resultant subunit structures of about 16 kDa and about 23 kDa via a S—S bond between the 161st cysteine and the 219th cysteine.
As an immunological method for measuring TRACP-5b, immunoassays using a polyclonal antibody and a monoclonal antibody are known (Non Patent Literature 5, Non Patent Literature 6, Non Patent Literature 7, Non Patent Literature 8, Non Patent Literature 9 and Non Patent Literature 10). Since TRACP-5a and TRACP-5b are collectively measured in these assays, the effect of TRACP-5a cannot be ignored (Non Patent Literature 11). Another immunological method for more specifically measuring TRACP-5b has been reported (Patent Literature 4). This method more specifically measures TRACP-5b activity; however, the antibody used in measurement is not specific to TRACP-5b and reacts also with TRACP-5a, resulting in that the activity is measured by taking advantage of difference in optimal pH between TRACP-5a and TRACP-5b and a measurement value was computationally obtained. Therefore, in a specimen from a patient with e.g., an end-stage renal disease in which TRACP-5a is enhanced, the effect is concerned. Furthermore, since the difference between a specimen of a healthy person and a specimen of a patient whose bone resorption is enhanced is small, sensitivity as a bone resorption marker is not sufficient (Non Patent Literature 12).
The present inventors previously formed a monoclonal antibody which can distinguishably recognize TRACP-5a and TRACP-5b (Patent Literature 6). As a result, they successfully formed an antibody having a certain selectivity; however, there is room for improvement in selectivity and a further improvement is required for clinical trials.